This invention relates to in vitro cell culture systems.
Given the appropriate conditions, many types of mammalian cells may be propagated in vitro. Typically, such conditions include a chemically-defined growth medium containing essential amino acids (e.g., cysteine, glutamine, and tyrosine), vitamins (e.g., biotin, choline, folic acid, thiamine), salts (e.g., NaCl, KCl, CaCl.sub.2), glucose, and, in many cases, serum. The role of serum is not completely understood, however it is purported to provide requisite trace materials, e.g., protein growth factors. In addition to a defined culture medium, many cultured mammalian cells require a solid surface on which to grow and divide. This requirement is most often provided by a glass or plastic tissue-culture dish or flask. In the absence of such a requirement, cells may be grown in suspension, e.g., in a test tube or tissue-culture bottle.
Two types of cultured mammalian cells are in widespread experimental and industrial use: "primary cells" and "immortalized cells". Primary cells are prepared directly from the normal tissues of an organism, e.g., from skin, bone marrow, or whole embryos. These cells grow well when first placed under culture conditions, typically surviving for weeks or months and dividing for about 50-100 generations. At that time, cells reach a stage, termed "crisis"; they grow and divide slowly, and soon, thereafter, cease to grow and divide altogether.
Immortalized cells are characterized by indefinite growth in vitro. Typically, immortalized cell cultures are derived either from a tumor sample explanted directly from an organism or from a primary cell variant which has undergone a change which promotes indefinite growth.
Hematopoietic cells may be propagated as primary cell cultures in vitro. Growth factors are provided, either by exogenous addition of the factors to the culture medium or by secretion of the factors by "feeder cells". Such feeder cells are generally stromal cells (i.e., a poorly defined mix of primary cells derived from bone marrow). A description of hematopoietic cell culture involving a stromal cell feeder layer is described by Whitlock et al. (J. Imm. Methods 67:353, 1984) and Whitlock and Witte (Meth. Enzymol 150:275, 1987); using their method, continuous cultures of early B-lymphocytes are established on an adherent feeder layer of dissociated mouse bone marrow cells. There is some support for the idea that hematopoietic cell growth also requires cell-cell contact. For example, Kincade et al. (Curr. Topics in Micro. & Imm. 135:1, 1987) report the dependence of cultured lymphoid cells on substances produced by, and/or on physical association with, an adherent stromal cell layer.